Voltage-gated potassium (Kv) channels are modulated by at least three distinct classes of accessory proteins including the KCNE family of single transmembrane proteins. In the human genome, KCNE proteins are encoded by five genes designated KCNE1 through KCNE5. The physiological importance of three KCNE genes has been demonstrated by associations with inherited disorders of cardiac and skeletal muscle excitability. Mutation of KCNE 1 impairs generation of the slowly activating cardiac delayed rectifier current (IKS), a critical potassium current contributing to myocardial repolarization that can be reconstituted in vitro by heterologous co-expression of recombinant KCNQ1 potassium channels with KCNE1. Other KCNE proteins also exert diverse functional effects on heterologously expressed KCNQ1. Co-expression of KCNQ1 with KCNE3, KCNE4 or KCNE5 is particularly interesting with functional effects ranging from constitutive activation (KCNE3) to suppression (KCNE4, KCNE5). We have recently demonstrated that all KCNE genes are expressed in human cardiac myocytes to varying degrees. We have further demonstrated that KCNE4 and KCNE5 exert potent inhibitory influences on heterologously expressed IKappaS and KCNE4 appears dominant over KCNE1 in this context. In addition to potential interactions with KCNQ1, KCNE proteins affect the functional properties of other cloned potassium channels. The physiological relevance of these in vitro observations remains uncertain. The observations that multiple KCNE proteins can modulate KCNQ1 channels to produce diverse biophysical phenotypes raises several intriguing questions with important implications for understanding the regulation of IKS as well as other potassium currents in normal and diseased heart. Can different KCNE subtypes compete functionally with KCNE1 for modulation of KCNQ1? Can KCNQ1 associate with more than one KCNE subtype in the same cell and at the same time? Do KCNE4 and KCNE5 participate in the pathophysiology of heart failure and other arrhythmia-prone conditions? In this proposal, we outline several experimental approaches for defining the physiological importance of KCNE subunits with a particular focus on KCNE4 and KCNE5. In Specific Aim 1, we will use a heterologous expression system to assess the range of functional and biochemical interactions possible between KCNQ1 and different KCNE subunits with a focus on KCNE4 and KCNE5. In Specific Aim 2, we will test the hypothesis that KCNE4 and KCNE5 are physiologically significant regulators of potassium currents in native cardiac myocytes by using two independent gene silencing approaches. Finally in Specific Aim 3, we will examine expression patterns of KCNQ1 and KCNE genes in diseased human heart and in an experimental canine model of ventricular arrhythmia susceptibility. Results from these studies will have implications for advancing our knowledge of the complex interplay between pore-forming and accessory subunits that comprise human potassium channels expressed in the heart and in other tissues.